Development of an improved standard reference material for folate vitamers in human serum

The US National Institute of Standards and Technology (NIST) developed a Standard Reference Material® (SRM®) 3949 Folate Vitamers in Frozen Human Serum to replace SRM 1955 Homocysteine and Folate in Human Serum. The presence of increased endogenous levels of folic acid and 5-methyltetrahydrofolate (5mTHF) in SRM 3949, enhanced folate stability via addition of ascorbic acid, and inclusion of values for additional minor folates are improvements over SRM 1955 that should better serve the clinical folate measurement community. The new SRM contains folates at three levels. To produce SRM 3949, pilot sera were collected from 15 individual donors, 5 of whom were given a 400-µg folic acid supplement 1 h prior to blood draw to increase serum levels of 5mTHF and folic acid for the high-level material. To stabilize the folates, 0.5% (mass concentration) ascorbic acid was added as soon as possible after preparation of serum. These pilot sera were screened for five folates plus the pyrazino-s-triazine derivative of 4-α-hydroxy-5-methyltetrahydrofolate (MeFox) at the US Centers for Disease Control and Prevention (CDC) by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Based on these results, a blending protocol was specified to obtain the three desired folate concentrations for SRM 3949. ID-LC-MS/MS analysis at the CDC and NIST was utilized to assign values for folic acid and 5mTHF, as well as several minor folates.